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Updated: Jul 11, 2025
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A Protocol for Using Förster Resonance Energy Transfer (FRET)-force Biosensors to Measure Mechanical Forces across the Nuclear LINC Complex
Published on: April 11, 2017
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Tunable force transduction through the Escherichia coli cell envelope
Daniel P Williams-Jones1, Melissa N Webby1, Cara E Press1
1Department of Biochemistry, University of Oxford, Oxford OX1 3QU, United Kingdom.
Summary
Gram-negative bacteria use proton motive force (PMF) to power outer membrane processes. This study reveals how PMF-driven motors generate mechanical force via transducer proteins, crucial for bacterial outer membrane stability and nutrient import.
Area of Science:
- Bacterial cell envelope biogenesis
- Membrane protein complexes
- Energy transduction mechanisms
Background:
- Gram-negative bacteria possess an outer membrane (OM) lacking intrinsic energy, necessitating inner membrane (IM) energy-transduction systems for OM processes.
- Tol-Pal and Ton systems, coupled to proton motive force (PMF), stabilize the OM and import nutrients, respectively.
- Both systems utilize IM motor complexes (homologous to flagellar stator Mot) to transduce force via IM proteins (TolA, TonB) to the OM.
Purpose of the Study:
- To elucidate the mechanism by which PMF-driven motors in the IM generate mechanical work at the OM through force transducers.
- To determine the structural basis of force transduction in the Tol-Pal system.
Main Methods:
- Cryoelectron microscopy (cryo-EM) to determine the 4.3Å structure of the Escherichia coli TolQR motor complex.
- In vivo assays using chimeric TolA/TonB proteins with swapped or replaced periplasm-spanning domains to probe force transduction mechanisms.
- Analysis of mutant TolA protein function in OM stabilization and colicin import.
Main Results:
- The cryo-EM structure of the TolQR motor complex reveals a 5:2 stoichiometry, consistent with related Ton and Mot complexes, supporting rotary motion.
- Chimeric protein assays demonstrate that structural rigidity of force transducer proteins, not specific structural forms, is key for efficient force conversion.
- TolA mutants display varying force outputs, correlating with their ability to stabilize the OM and import colicins.
Conclusions:
- The study provides structural insights into the TolQR motor complex, supporting a rotary mechanism for PMF-driven force generation.
- Efficient conversion of rotary motion into physiologically relevant force at the OM is primarily governed by the structural rigidity of force transducer proteins like TolA.
- This mechanism is critical for essential bacterial outer membrane functions, including stability and nutrient uptake.
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